As biofunctional molecules, peptides and proteins are used ubiquitously as essential reagents in basic life science research. More importantly, many bioactive peptides and proteins also have direct therapeutic value. In fact, peptide- and protein-based biologics are a mainstay therapeutic modality of modern day pharmaceutical industry, and about 40% of all experimental drugs currently under the various stages of clinical development are peptides and proteins, or their bioconjugates. Therefore, methods that allow for efficient synthesis of these biomolecules not only can provide important enabling technologies for basic biomedical research, but also for drug discovery.
Traditionally, solid phase peptide synthesis (SPPS) is the method of choice for preparing small to medium-sized peptides. Since its discovery, SPPS has been a choice of use in the synthesis of countless peptides. As an advantage to the chemical synthesis, peptides synthesized by SPPS may contain any kinds of structural units, such as D-amino acids and other non-natural amino acids, and/or have some unusual architectures. However, for practical reasons, it is technically very challenging to use SPPS for synthesizing very large peptides or proteins. Recombinant DNA technology is the classic method for protein production. However, this technology suffers from several limitations, such as inability to produce unnatural or post-translationally modified proteins, difficulty in expressing multi-domain proteins or proteins that are toxic to the host cell, and problems of product heterogeneity due to uncontrolled processing of nascent polypeptide chains.
A significant advance in chemical biology for the past two decades has been the development of chemoselective peptide ligation methods for protein synthesis. With these advance methods, one can use peptides and protein domains produced by SPPS and recombinant DNA technology as building blocks for the construction of large, complex protein molecules, therefore overcoming the limitations of existing technologies. As a common feature, almost all the currently available ligation chemistry is characterized by a typical two-step reaction scheme: a prior capture step to bring together the reacting C- and N-termini of two peptide components, followed by an intra-molecular reaction for peptide bond formation.
For many of these ligation methods, the capture reaction usually involves a side-chain functional group on the N-terminal amino acid of the second peptide component. For instance, the unique soft nucleophilicity of the thiol group has been exploited as the capture device in the development of the thioester-cysteine ligation method (also known as native chemical ligation) whereby the capture step is a transthioesterification reaction between a C-terminal thioester of the first peptide and the N-terminal cysteine (Cys) thiol group of the second peptide. This ligation method has also been extended to non-Cys residues through introducing a temporary thiol group onto their side chains. Similarly, the thioacid capture ligation method is so-named because a super-nucleophilic C-terminal thioacid of the first peptide can be captured very efficiently by the Npys-activated thiol of the N-terminal Cys residue of the second peptide. The 1,2-aminoethanethiol of N-terminal Cys and 1,2-aminoethanol moiety of an N-terminal Ser/Thr residue have also been used to develop the so-called aldehyde capture ligation methods. In fact, the glycoaldehyde ester-mediated ligation is the earliest peptide ligation method developed, in which a highly selective thiazolidine/oxazolidine formation reaction serves as the capture reaction which is followed by an S- or O-to-N acyl transfer step to form a pseudoproline residue at the ligation site.
Recently, a C-terminal salicylaldehyde ester is used to replace the glycoaldehyde ester to give a highly efficient ligation reaction after which the salicylaldehyde moiety can be readily removed in an acidolysis reaction to generate native Ser or Thr at the ligation junction. This development is significant in that it is one of the rare methods that does not depend on a thiol group for the ligation reaction and that Ser or Thr is much more abundant than Cys in natural proteins. Other ligation methods that do not depend on a sidechain thiol or any sidechain functionality include the Staudinger ligation and the decarboxylative condensation method. However, these two methods have limited practical utility because of the low efficiency of the ligation reaction.
As seen from above, so far all the existing methods require a side-chain functional group on the N-terminal aminoacid residue of the second peptide to mediate the ligation reaction, which limits the application scope of these methods.